Resolution of the effects of sulfhydryl-blocking reagents on hormone-and DNA-binding activities of the chick oviduct progesterone receptor

The differential effects of sulfhydryl (SH)-blocking agents on hormone and DNA binding by the chick oviduct progesterone receptor were investigated. Previous studies have demonstrated inhibition of steroid-receptor interaction by SH-blocking agents and protection against inhibition by bound hormone. The present results indicate that the SH group required for steroid binding is within or near the hormone-binding site itself, and that a second SH group (or groups) is involved in the binding of receptor to DNA. Three findings relate to the site of action of SH-blocking agents on hormone binding. First, glycerol decreased the rate of hormone dissociation and the rate of hormone displacement by mercurial reagents by 75 to 90%. Second, mercurial reagents displaced [³H]progesterone bound to the mero-receptor, a M_r 23,000 proteolytic fragment containing the hormone-binding site, but not the site of interaction with DNA. Third, hormone displacement was still present after a 10,000-fold purification of the progesterone receptor. Mercurial reagents also inhibited binding of progesterone receptor to DNA, whereas the SH-alkylating agents N-ethylmaleimide and iodoacetamide had no effect. It is likely that distinct sulfhydryl groups are required for steroid receptor interaction with hormone and with DNA, since brief treatment with mercurial reagents blocked DNA binding, but caused only a slight displacement of bound hormone. The SH group required for hormone binding probably lies within or near the hormone-binding site, is sensitive to mercurials, alkylating agents, and 5,5′-dithiobis(2-nitrobenzoate) (DTNB), and is protected by bound hormone. The SH group required for DNA binding, in contrast, is sensitive to mercurials but not to alkylating agents, is only partially sensitive to DTNB, and is not protected by bound hormone.     

Changes in adenylate cyclase and phosphodiesterase activities during the growth cycle of adult rat hepatocytes in primary culture

Long-term primary adult rat hepatocyte cultures show growth-state-dependent changes in adenylate cyclase and cAMP phosphodiesterase activities. Cellular adenylate cyclase activity decreases to undetectable levels within 1 day postplating, reappears on Days 4-5, and becomes maximal on Day 9. Membrane adenylate cyclase and cellular cAMP formation are insensitive to glucagon during log phase (Days 4-8) but not during lag (Day 1) or stationary phase (Day 12). Cyclic AMP phosphodiesterase activities (soluble and particulate) fall approximately equal to 70% by Day 2 but recover as proliferation begins. By contrast, the particulate phosphodiesterase assayed at 100 microM cAMP, decreased during Days 0-2. These observations simulate changes seen during liver proliferative transitions in vivo and, therefore, further support the use of these cultures as a developmental model.     

GABA modulation of cholinergic transmission in rat oviduct

The effects of electrical stimulation, γ-aminobutyric acid (GABA), acetylcholine (ACh), norepinephrine (NE), 5-hydroxytryptamine (5-HT), GABA agonists and bicuculline were studied on spontaneous movements of isolated rat oviduct. The tissue did not respond to electrical stimulation or to GABA, NE and 5-HT when added to the incubation medium. ACh produced contractions related to its concentration which were maximal at the diestrous-1 phase when GABA caused a 20% rise in the ACh contraction. This effect was mimicked by GABA agonists whereas it was suppressed by bicuculline. β-Estradiol benzoate (EB) increased ACh contractions in diestrous-1 and in the late proestrous phases. GABA did not modify the EB effect. Progesterone did not modify ACh contractions in any of the studied phases. These findings suggest a possible modulatory role for GABA on ACh responses in the isolated rat oviduct.     

Differential expression of aortic and lung elastin genes during chick embryogenesis

The ratios of tropoelastin b to a were measured in chick aorta and lung during embryogenesis. The rates of tropoelastin a and b synthesis were determined in short-term organ culture. The results demonstrated that in lung tissue the ratio of the two tropoelastins remained essentially constant. Each of the tropoelastins comprised 50% of the total elastin synthesis. In the aortic tissue, tropoelastin b represented 70% of the total elastin in the 11- to 13-day embryos and increased to 91% by Day 16. These observations seen in the organ culture system were paralleled in measurements of functional mRNAs coding for the two proteins. Measurements of functional tropoelastin mRNAs from both lung and aortic tissues were performed in a mRNA-dependent rabbit reticulocyte lysate system. Although the changes in the abundance of the tropoelastin mRNAs revealed the same trend as that seen in the organ culture data, the magnitude of the tropoelastin b to a ratio in the aortic organ culture was twice that determined in the cell-free translation of aortic mRNAs. The data obtained from both cell-free translations and organ culture experiments demonstrate that there is a differential expression of elastin genes during aorta development which is significantly different from that found in developing lung.     

The onset of activation responsiveness during maturation coincides with the formation of the cortical endoplasmic reticulum in oocytes of Xenopus laevis

Immature, Stage VI oocytes of Xenopus laevis fail to activate (i.e., to propagate a cortical reaction and elevate a fertilization envelope) when pricked or exposed to A23187. We determined the times during maturation when immature oocytes treated with progesterone in vitro developed the capacity to respond to pricking and to ionophore. Responsiveness to ionophore first appears at about 3.5-4.5 hr after progesterone treatment; all oocytes are activated by 8-9 hr after progesterone. The capacity to respond to pricking appears about 1.0-1.5 hr after first signs of ionophore responsiveness. We examined the cortical endoplasmic reticulum (CER) by TEM to determine whether the morphology of this component could be correlated with the development of responsiveness during maturation. Fully mature oocytes exhibit an extensive CER that (1) forms a "shell" around most cortical granules, (2) appears to interconnect cortical granules, and (3) forms junctions with the plasma membrane. The CER-plasma membrane junctions are especially obvious in preparations of isolated cortex. The elaborate CER is not present in immature oocytes. It first appears during maturation of progesterone-treated oocytes at 4.5-5.0 hr, coincident with the time when maturing oocytes develop their responsiveness to ionophore and to pricking. This temporal correlation is consistent with the hypothesis that the CER is one of the components required for regulation of intracellular free calcium in oocytes.     

Changes in transferrin during the red cell replacement in amphibia

Transferrin, a plasma glycoprotein, carries iron from storage sites to immature erythroid cells for hemoglobin synthesis. The replacement of larval red cells by adult red cells, which occurs during metamorphosis in bullfrogs, requires extensive formation of hemoglobin and new red cells. Large changes in red cell iron storage also occur during the red cell replacement. Both the concentration and the level of iron saturation of plasma transferrin were measured during metamorphosis to determine if there were changes in plasma transferrin which coincided with the changes in red cell iron storage and ferritin content. Plasma transferrin concentrations increased from 0.96 to 2.6 mg/ml during the period when red cell storage iron and ferritin decreased. Plasma iron concentrations also increased when the transferrin concentration increased, suggesting that the additional transferrin may be involved in moving iron from the larval red cell stores. At the end of metamorphosis, the plasma iron concentration decreased to premetamorphic levels but the transferrin concentration remained high, resulting in a decrease in saturation to 18% compared to 45% in the larvae. In addition to differences in iron saturation, adult transferrin had different electrophoretic properties from larval transferrin. The results support the hypotheses that during early ontogeny plasma transferrin and red cell iron storage are coordinated to provide iron for the formation of the first generation of adult red cells and that transferrin may participate in the control of red cell ferritin synthesis.     

Effects of medium composition and metabolic inhibitors on glycosaminoglycan synthesis in chick embryo cartilage and its stimulation by serum and triiodothyronine

Incorporation of inorganic sulfate into glycosaminoglycans of chick embryo sternum is stimulated by serum and triiodothyronine. Variations in the amino acid content of the medium, and in particular in the concentration of glutamine, changed the incorporation in control and stimulated sterna to the same degree. Omission of Na⁺ from the medium greatly reduced incorporation in both control and stimulated sterna; incorporation, and its stimulation by triiodothyronine, was restored by raising the concentration of Na⁺. Ouabain and valinomycin inhibited incorporation more than 90%, and triiodothyronine did not stimulate under these conditions. Puromycin and cycloheximide also inhibited incorporation almost completely, and abolished the stimulation by triiodothyronine and serum. Addition of p-nitrophenyl-β-xyloside, in the presence of puromycin or cycloheximide, restored sulfation to a level of 5–10% of the control value; however, this level of incorporation was not increased by addition of serum or triiodothyronine.Actinomycin D, colchicine and vinblastine inhibited incorporation by 40% or less at the highest concentrations tested; however, these three agents completely abolished the ability of triiodothyronine to stimulate incorporation. Lumicolchicine and cytochalasin B decreased incorporation in controls slightly, but did not affect the stimulation by serum or triiodothyronine.The results indicate that thyroid hormones stimulate glycosaminoglycan synthesis only under conditions which support efficient synthesis in control incubations, and suggest that microtubule formation may be essential to the mode of action of thyroid hormones in this system.     

Changes in DNA-polymerase and thymidine kinase activity during interferon treatment

Treatment of the human glioma cell line, U-251 MG, with human IFN-β resulted in a dose-dependent growth depression and a decreased activity of DNA-polymerase in exponentially growing cells, although paradoxally the number of cells in the S phase increased. In synchronized cells, a S block was confirmed. Both thymidine kinase and DNA-polymerase increased but with a lower rate during IFN treatment. No inhibitory effects on any of the enzymes could be seen when IFN-treated lysate was mixed with control lysate. The possible significance of depressed DNA synthesis during virus infection is discussed.     

Changes in the activities of the protoheme-synthesizing system during the growth of yeast under different conditions.

The levels of some enzymatic activities involved in protoheme synthesis have been measured in subcellular fractions obtained at different stages of the growth of the yeast Saccharomyces cerevisiae grown anaerobically and aerobically with glucose (50 or 6 g/ liter), and ethanol (20 g/liter) as the carbon source. The degree of repression of the respiratory system is estimated by the respiratory capacity of whole cells, by the activities of succinate-cytochrome c reductase and cytochrome c oxidase of the mitochondrial particles, and by the cytochrome spectra. The results show that (i) the more porphyrins (cytochromes) that are synthesized by the cells, the lower is the specific activity of δ-aminolevulinic acid (ALA) synthetase and the higher is the specific activity of ALA dehydratase, the activity ratio ALA synthetase/ALA dehydratase decreasing at least 10-fold compared to the repressed cells; (ii) the amount of intracellular ALA found under all conditions tested (from 0.05 to 1.5 mm in the cell sap) correlates well with the measured ALA synthetase activity; its presence argues against a rate-limiting function for ALA synthetase and rather favors such a role for the ALA dehydratase in the formation of heme in yeast; (iii) the rate of porphyrin synthesis measured in vitro is higher in the case of cells with high cytochrome contents; and (iv) the specific activities of succinyl CoA synthetase and protoheme ferrolyase are always present in nonlimiting amounts. Some experiments are described showing that the values of the activities which are calculated from these in situ and in vivo experiments compare well with the values measured in vitro in the acellular extracts. The results concerning the enzymatic activities, together with (i) the excretion of coproporphyrin(ogen) and the accumulation of protoporphyrin + Zn-protoporphyrin in anaerobiosis, (ii) the presence of protoporpho(di)methene (P503) in anaerobic and repressed cells, and (iii) the presence of intracellular ALA under all growth conditions, are discussed in terms of possible control(s) of heme synthesis in yeast.     

Choline acetyl transferase activity in chick embryo neuroretinas during development in ovo and in monolayer cultures

The specific activity of the enzyme choline acetyl transferase (CAT) in chick neuroretinas was investigated during in ovo development and in monolayer cultures. The enzyme activity was barely detectable on the 6th day of incubation but increased markedly between the 7th and 11th days. The activity increased sharply between the 15th and 17th days and then slowly until hatching. When cell suspensions from 6- to 7-day neuroretinas were cultured as monolayers, CAT specific activity increased rapidly. After 4–5 days in culture, the activity of the enzyme was identical to that found in the neuroretina on the 11th day of incubation. Cells from 9-day neuroretinas also differentiate in monolayer cultures, but with a more irregular pattern. These data show that cholinergic neurons from chick embryo neuroretina differentiate in monolayer cultures without a lag and at the same rate as in vivo.     

Augmentation of B-cell responsiveness by a tumor-activated T-cell factor

The growth of the P815 mastocytoma in syngeneic DBA/2 mice led to an activation of Ly1+2- T cells. These T cells produced a soluble factor or factors in culture which, when added to normal spleen cells or B cells in the presence of syngeneic Ly1 cells, caused a genetically unrestricted augmentation of the plaque-forming response toward sheep red blood cells (SRBC). The culture supernatant of the activated T cells did not support the proliferation of an interleukin-2 (IL-2)-dependent cell, nor exhibit properties of late-acting TRF. Active supernatants appeared to affect directly B cells during the first 48 hr of culture with SRBC in such a way as to make them more responsive to antigen-specific Ly1-cell help.     

Epitopes in human growth hormone and chorionic somatomammotropin studied with monoclonal antibodies

The immune complexes formed by human growth hormone or human chorionic somatomammotropin and various monoclonal antibodies have been studied by gel filtration and polyacrylamide gel electrophoresis. Two of the monoclonal antibodies gave rise to complexes with molecular weights suggesting an antigen:antibody 1:1 ratio. When both antibodies were simultaneously incubated with human growth hormone the ratio estimated for the new complex was 1:2, indicating the existence of two nonoverlapping epitopes in the antigen. The other monoclonal antibodies exhibited a more intricate behavior: incubated separately with human growth hormone they gave rise to both types of the aforementioned complexes. A similar phenomenon could be demonstrated with human chorionic somatomammotropin. The study of the immunoreactivity of a synthetic peptide indicates that the involved epitopes are localized within the region limited by amino acid residues 44 and 128 of human growth hormone.     

Identification of two new collagen alpha-chains in extracts of lathyritic chick embryo tendons

Two new collagen polypeptide chains have been identified in extracts of lathyritic embryonic chick tendons. The electrophoretic migration of these polypeptides in sodium dodecyl sulfate-polyacrylamide gels indicates that they have about 20% greater apparent molecular weights than α₁ and α₂ chains of Type I collagen. These chains are not held by disulfide bonds since reduction does not affect their electrophoretic behavior. Further, they do not represent incompletely cleaved procollagen since their apparent molecular size remains greater than that of Type I collagen polypeptides after limited proteolytic digestion. Because the ratio of these polypeptides in the purified extracts is not 2:1 it appears that they are components of two separate tropocollagen molecules.     

Changes in the post-translational modification of lysosomal enzymes during development of Dictyostelium

The form of post-translational modification present on two lysosomal enzymes--acid phosphatase and alpha-mannosidase--changes as part of the developmental program of Dictyostelium discoideum. Prior to 8 h of development, all enzyme molecules are of a single modification type (early form enzyme). Starting at 8 h of development, enzyme molecules with a second type of modification (late-form enzymes) begin to appear in the cell. We separated the early and late forms of these enzymes from each other by chromatography on DEAE-cellulose. We found that the change in protein modification affects the enzymes' in vitro properties. The early and late forms of both of these enzymes differ in thermostability and susceptibility to proteolytic inactivation. We also found that the late form of alpha-mannosidase is preferentially secreted. We suggest that by synthesizing molecules with a second form of modification, the cell confers new characteristics to its lysosomal enzymes.